This document discusses various enzymes that are used in genetic engineering and recombinant DNA technology. It describes DNA and RNA polymerases such as DNA polymerase I, Klenow fragment, T4 DNA polymerase, and reverse transcriptase. It also covers ligases, phosphatases, kinases, and nucleases including DNase I, and their functions, sources, and applications in techniques like cDNA synthesis, DNA labeling, amplification, and sequencing.

1. ENZYMES USED
IN
GENETIC ENGINEERING-I
1
Lecture- 10

2. Several enzymes are used in recombinant DNA technology for
carrying out various modifications of nucleic acids.
DNA polymerase I enzyme, first isolated from E. coli by Arthur
Kornberg and coworkers in 1958, is used in synthesis of second
strand of cDNA (copy or complemenatary DNA). It is also used in
the nick translation technique for radiolabelling of DNA.
David Baltimore (1970) and Howard Temin and Satoshi Mizutani
(1970) independently isolated RNA-dependent DNA polymerase
(reverse transcriptase) enzyme from the virions of RNA tumour
and Rous sarcoma viruses, respectively. This enzyme is used for
the synthesis of first strand of cDNA from an RNA template.
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3. DNA POLYMERASES
DNA POLYMERASE I
 KLENOW FRAGMENT
 T4 DNA POLYMERASE
 THERMOSTABLE DNA POLYMERASES
 TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE
3

4. DNA Polymerase I
SOURCE
E.coli
FUNCTION
• 5’ 3’ Exonuclease activity
• 3’ 5’ Exonuclease activity
• 5’ 3’ Polymerase activity : Addition of dNTP’s at 3’-OH
termini of DNA/RNA primers.
• Fills gaps in ds DNA
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5. APPLICATIONS
• Synthesis of second strand of cDNA.
• Preparation of Radioactive Probes by end-labelling of DNA
• DNA labeling by Nick Translation
NICK
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6. KLENOW FRAGMENT
SOURCE
DNA Polymerase I treated with protease subtilisin
FUNCTION
•DNA Polymerase I without 5’ 3’ Exonuclese activity is called
Klenow Fragment.
• Has 3’ 5’ Exonuclese activity, 5’ 3’ Polymerase activity.
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7. APPLICATIONS
• Synthesis of dsDNA from single stranded templates
• Filling in recessed 3’ ends of DNA fragments
• Digestion of protruding 3’ overhangs.
• Preparation of radioactive probes by end-labelling of DNA
• Random primer labeling of DNA
• In-vitro mutagenesis using synthetic oligonucleotides
• DNA sequencing by di-deoxy chain termination method
• DNA Amplification
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8. FILLING OF 3’ RECESSED END BY KLENOW FRAGMENTOF E.coli DNA POLYMERASEI
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9. Functional domains in the Klenow Fragment (left) and DNA
Polymerase I (right). 9

10. T4 DNA Polymerase
SOURCE
Encoded by T4 bacteriophage
FUNCTION
• Requires primed single stranded template.
• Has 3’ 5’ Exonuclease activity, 5’ 3’ Polymerase activity.
• Catalyze template directed DNA synthesis from free 3’-OH
end bound to primer.
• High processivity (400 nucleotides/second)
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11. APPLICATIONS
• Filling in recessed 3’- ends of DNA fragments
•Preparation of radioactive probes by end-labelling of DNA
• End labeling of recessed 3’-ends
• End labeling of protruding 3’-ends
• Labeling DNA fragments for use as hybridization probes
• Conversion of cohesive ends of duplex DNA into blunt-end
DNA
• In-vitro mutagenesis using synthetic oligonucleotides
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12. T7 DNA Polymerase
SOURCE
Synthesized from E.coli infected with T7 bacteriophage
FUNCTIONS
• Has 3’ 5’ Exonuclease activity, 5’ 3’ Polymerase activity.
•Highprocessivity than other thermolabile bacterial DNA
polymerases
APPLICATIONS
•End-labeling
•Extension of primer
•DNA Sequencing
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13. THERMOSTABLE DNA POLYMERASES
SOURCE
Thermophilic and Hyperthermophilic eubacteria and
thermophilic archea
First thermostable DNA polymerase was isolated and
characterized from Thermus aquaticus
FUNCTION
• Catalyze template directed DNA synthesis from free 3’-OH
end bound to primer.
APPLICATION
• In-vitro DNA amplification by PCR
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14. TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TdT)
SOURCE
•Immature, pre-B, pre-T lymphoid cells and acute
lymphoblastic leukemia/lymphoma cells
•Commercially available TdT is purified from recombinant E.
coli cells expressing calf / rat / mouse thymus gene.
FUNCTION
•Adds a particular nucleotide to the 3’-end of a DNA strand
• Does not require a template
• Preferred substrate is protruding 3’ overhang.
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15. APPLICATIONS
• Probe preparation
• Cloning by homopolymer tailing
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16. REVERSE TRANSCRIPTASE
SOURCE
RETROVIRUSES
• Moloney murine leukemia virus (Mo-MLV)
• Avian myeloblastosis virus (AMV)
FUNCTIONS
RTases have two types of activities:
DNA Polymerase Activity
Transcribes both ssRNA and ssDNA templates by using RNA
and DNA primers respectively.
RNaseH activity
Functions as both ENDONUCLEASE and EXONUCLEASE.
Degrades RNA in RNA:DNA hybrid, formed during reverse
transcription of an RNA template.
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17. APPLICATIONS
• In-vitro reverse transcripton of mRNA
• Reverse transcription PCR
• Labeling of DNA molecule
• Sequencing of DNA
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18. RNA Polymerase
E.coli RNA Polymerase
•Multisubunit enzyme of E.coli
•DNA dependent RNA polymerase
•Makes RNA copy of DNA/RNA
Bacteriophage RNA Polymerase
SOURCE
Purified from phage (e.g. SP6, T7, T3) infected bacteria or
produced as recombinant proteins
FUNCTION
•DNA dependent RNA polymerase
•High specificity for double stranded promoters
•Catalyzes 5’ 3’ synthesis of RNA using either ssDNA/dsDNA
as template
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19. APPLICATIONS
• Synthesis of ssRNA transcripts
• Expression of cloned gene into bacteria
• In-vitro synthesis of capped RNA transcripts
• RNase protection assays
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20. PolyA POLYMERASE
SOURCE
Recombinant protein isolated from E. coli
FUNCTION
Template independent polyadenylation at 3’-terminus of
RNAs
APPLICATIONS
• Production of poly-A tailed RNA
• 3’-end labeling of RNA
• Determination of polyA+ RNA content
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21. ALKALINE PHOSPHATASE
SOURCE
Bacterial alkaline phosphatase
Calf alkaline phosphatase
Arctic Shrimp Alkaline phosphatase
FUNCTION
• Removal of 5’-phosphate groups from DNA and RNA
• Acts on 5’-overhangs, 5’-recessed ends, Blunt ends
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22. APPLICATIONS
• Prevention of self ligation of vector
• Removal of 5’ Phosphate group before end labeling
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23. POLYNUCLEOTIDE KINASE (PNK)
SOURCE
Bacteriophage pseT gene expressed in E. coli
FUNCTION
• Transfers у- phosphate from ATP to the 5’-end of DNA/RNA
• PNK also has 3’ phosphatase and 2’,3’ cyclic
phosphodiesterase activities , although of little significance
APPLICATIONS
• Phosphorylation of polynucleotide
• Radiolabeling of 5’ - termini
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DNA Ligase
Apart from cutting of DNA, another major
requirement of recombinant DNA technology is the joining
of DNA fragments.
•This is done by using DNA ligase enzymes isolated from
E. coli or bacteriophage T4-infected E. coli bacteria. In the
beginning of 1967 Martin Gellert reported the formation of
covalent circles of bacteriophage lambda DNA by using an
E. coli cell extract.
•Towards the end of 1967, four research groups, including
that of Martin Gellert, independently isolated DNA ligase
enzyme.

26.  BACTERIOPHAGE T4 DNA LIGASE
 E. coli DNA LIGASE
 Taq DNA LIGASE
TYPES OF DNA LIGASES
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28. BACTERIOPHAGE T4 DNA LIGASE
SOURCE
T4 bacteriophage infected E. coli
FUNCTION
•Most commonly used for DNA ligations
• Catalyzes formation of phosphodiester bonds between
juxtaposed 5’-phosphate and 3’-OH ends in DNA (cohesive
ends)
• Repairs single stranded nicks in dsDNA
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29. APPLICATIONS
• Ligation of cohesive ends
• Ligation of blunt ended termini
• Ligation of synthetic linkers or adaptors
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30. E. coli DNA LIGASE
SOURCE
E. coli
FUNCTION
• Catalyzes formation of phosphodiester bonds in dsDNA
containing cohesive ends
• In some cases, catalyzes blunt end ligation also (in presence
of PEG)
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31. APPLICATIONS
• Ligation of cohesive ends
• Cloning of full length cDNA
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32. Taq DNA LIGASE
SOURCE
Thermus aquaticus
FUNCTION
• Catalyzes joining of nicks in dsDNA
• Also catalyzes blunt end ligation at elevated temperatures,
in presence of certain agents
APPLICATIONS
•Detection of mutation
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33. DEOXYRIBONUCLEASE (DNase)
 DNaseI
 STAPHYLOCOCCAL NUCLEASE
 SHRIMP DNase
 S1 NUCLEASE
 MUNGBEAN ENDONUCLEASE
 Bal31 NUCLEASE
 EXO-DEOXYRIBONUCLEASES
EXONUCLEASE I
EXONUCLEASE III
EXONUCLEASE V
EXONUCLEASE V (Rec B,C,D)
λ- EXONUCLEASE
T7 GENE6 EXONUCLEASE
TYPES
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34. DNase I
SOURCE
Bovine Pancreas
FUNCTION
• Endonuclease that catalyzes degradation of both ss and ds
DNA into di-, tri-, and oligonucleotides with 5’-phosphate and
3’-hydroxylated termini
•Acts on ss and ds DNA and RNA:DNA hybrids
• Randomly produces nicks independently into each dsDNA in
presence of Mg2+
APPLICATIONS
• Removal of DNA contamination
• Labeling of DNA by NICK-TRANSLATION
• DNase I footprinting
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